A TWO-PHOTON HOLOGRAPHIC MESOSCOPE FOR HIGH-RESOLUTION OPTOGENETIC PHOTOSTIMULATION AT THE MILLIMETER SCALE

Two-photon (2P) all-optical approaches enable the simultaneous imaging and manipulation of neuronal activity with single-cell resolution, providing powerful tools to probe the causal principles of neural circuit function in vivo. However, despite major advances in large-field 2P imaging, the application of patterned 2P photostimulation over millimeter-scale fields of view (FOV) remains limited. In particular, holographic approaches based on spatial light modulators (SLMs) are constrained by optical configuration, wavelength dispersion, and geometric trade-offs that restrict the achievable stimulation FOV while preserving cellular-scale resolution.
Here we present a 2P holographic mesoscope (2P-HOLMES) that combines computer-generated holography with a commercial SLM and optical fiber bundles. By exploiting the high divergence and inter-core delay dispersion of fiber bundles, this approach enables millimeter-scale photostimulation FOVs while maintaining near single-cell lateral and axial confinement. We challenge the fundamental limits of conventional holographic configurations and show, through optical simulations and experimental characterization, that 2P-HOLMES achieves axial resolutions below 35 µm across a 3 mm FOV, substantially exceeding current 2P holographic approaches.
We validate the system in vivo by performing large-FOV 2P calcium imaging together with patterned optogenetic photostimulation in mouse cortex and cerebellum, enabling the simultaneous targeting of widely separated neuronal populations. By extending holographic photostimulation to mesoscopic scales, 2P-HOLMES enables flexible, reconfigurable, and scalable all-optical interrogation of distributed neural circuits, opening new opportunities to study long-range interactions with cellular precision.